Project Summary/Abstract A crucial step in the development of human cancer is the ability of cells to undergo immortalization by avoiding senescence and apoptosis that result from repeated cell divisions. To accomplish this, 90% of cancers reactivate telomerase reverse transcriptase (TERT), the catalytically active and rate-limiting subunit of telomerase. Telomerase functions to maintain telomeres, which cap the ends of chromosomes, protecting chromosomal DNA from the end replication problem. Normally, TERT is expressed in stem cells, but is transcriptionally silenced in somatic cells. However, recent work has demonstrated that many cancer subtypes, including 83% of primary glioblastoma (GBM) tumors, contain activating mutations in the TERT promoter. These mutations result in binding of the transcription factor GA binding protein (GABP), and reactivation of TERT, through incompletely understood mechanisms. Understanding and targeting genes and pathways related to these phenomena has the potential to open major future therapeutic avenues for GBM, the most common and most severe form of adult brain cancer. The current proposal will make use of a focused in vivo CRISPRi growth-based screen using an intracranial xenograft model of GBM to uncover factors that regulate immortality of TERT promoter mutant GBM cells. The targeted screen will be performed in both control and GABP mutant cell lines, in order to elucidate genes that function both synergistically with and independently of GABP. In parallel, the proposal aims to engineer Cas9 ribonucleoprotein (RNP) complexes that target GBM cells specifically, using receptor-mediated uptake. Cas9 RNP complexes are currently being developed with future therapeutic intent, given the tendency for reduced toxicity and off-target effects as compared to other methods of CRISPR editing. These GBM-specific RNPs will be designed with the ultimate goal of editing genes to reverse cellular immortality in TERT promoter mutant GBM cells, both in vitro and in vivo. Overall, this work will develop CRISPR-based technologies for both in vivo screening and targeting of GBM cells, with the goal of elucidating and inhibiting major factors that underlie GBM cellular immortality.